EDC Impressum & DSE

Electrophoresis
Development & Consulting
Dr. Hanspeter Schickle
Wolfgang Gstrein

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Too much fat in the sample

Mai 30, 2018

Precipitating material at the anodal slot-edge, channal forming (See Track 2)

Too much fat

1) Too much fat in the sample

Remedy #1: Use special extraction buffer

Do not extract your proteins directly from the raw materials using detergent (SDS) -containing buffers!
Correct extraction procedure:

Extraction buffer:
0.25 M TRIS, 0.2M Glycine, 0.001 M EDTA (pH 9.1), detergent max. 0.05%

Extraction procedure:
15 min ultra sonic treatment - 10 min centrifugation 10 000 rpm - take the supernatant!

Remedy #2: Defatting the samples with Kerosene (simple procedure), without sample concentration

Add (~10% of the sample volume) Kerosene (stained with Sudan-red) and shake well. Leave 5 min to separate the Kerosene from the aqueous phase. Do not take the red hydrophobic phase.

Then add SDS, the reducing agent (DTT) and heat 3 - 5 minutes to 95 °C

Remedy #3: Acetone-precipitation with optional sample-concentrating

Precipitate the proteins with 100% Acetone. Cooling, then centrifuge samples.
Resolubilize with sample buffer, + heating 3-5 min 95°.

Defatting and desalting with optional concentration of the samples

Action

Remarks

Time

precipitation with 100% Acetone

50 µl sample + 200 µl 100% Acetone

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cooling at -20°C

Cool down to precipitate completely at (-20°C)

60 min

centrifugation

15 000 rpm, then discard the supernatant

10 min

drying

open the cups to remove the Acetone

30 min

resuspensation / concentration

add (fe.) 10 - 100 µl sample buffer (2% SDS)

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heating the samples

95°C: will charge the proteins with SDS

3-5 min